P-SELECTIN-MEDIATED AND CD18-MEDIATED RECRUITMENT OF CANINE NEUTROPHILS UNDER CONDITIONS OF SHEAR-STRESS

Neutrophil mobilization at sites of inflammation or thrombosis involve s the participation of several adhesion molecules expressed on neutrop hils and vascular endothelial cells. Local vascular damage with disrup tion of the endothelium results in adhesion of platelets to the expose d subendothelium, and these platelets could also participate in neutro phil recruitment. This initial phase of mobilization could be followed by heterotypic aggregation to recruit more leukocytes in the area. Th e present study first examined the interactions of adherent canine pla telets and flowing canine neutrophils using an in vitro system that si mulates vascular flow conditions. Results showed that collagen-adheren t platelets express the adhesion molecule P-selectin on their surface and can support neutrophil arrest (612 +/- 43 neutrophils/mm(2)) at sh ear stresses of approximately 2.5 dynes/cm(2). Both transient adhesion (manifested by a rolling-type behavior) and complete arrest were obse rved. These interactions could be totally inhibited by a monoclonal an tibody directed against platelet P-selectin (24 +/- 18 neutrophils/mm( 2)) but not by a monoclonal antibody against neutrophil CD18 (625 +/- 46 neutrophils/mm(2)). Additionally, under shear mixing conditions (70 0 RPM), canine blood leukocytes exhibited aggregation (>80% singlets r ecruited into aggregates after 5 minutes), and this process does not i nvolve P-selectin but is dependent on the neutrophil integrin CD18. Ho wever, stimulation of the blood with platelet-activating factor (5-20 ng/ml) induced a rapid aggregation with a significantly greater number of aggregates when compared with stirring alone (68.3% +/- 3.2% versu s 35.2% +/- 6.3% at 1 minute, P < 0.05), and this aggregation was both P-selectin and CD18 dependent. Overall, these two mechanisms of leuko cyte recruitment (neutrophil arrest on adherent platelets and aggregat ion) could act sequentially and in a cooperative manner to bring into close contact platelets and neutrophils at sites of inflammation and t hrombosis in pathologic conditions in the dog.